A number of eukaryotic transcription units have been shown to contain multiple polyadenylation sites that are used with different frequencies at different times. One such regulated unit is the adenovirus major late transcription unit. Early in infection, the L1 poly(A) site is used more frequently than the L2 site, whereas late in infection the L2 site is chosen more often. The adenovirus system is a useful system because the virus depends upon the ce 1's transcriptional machinery; thus, the results obtained are applicable to the study of cellular genes. The aim of this proposal is to characterize the sequence requirements of these two poly(A) sites and ultimately study their regulation in vitro. To accomplish this, the two poly(A) sites will be cloned and mutagenized. They will first be assayed in transfections to define the sequences necessary for polyadenylation. It will also be determined by transfection into infected cells if there are distinct sequence requirements for the regulation of poly(A) site choice. The affect of changing the relative positions of the two sites will also be studied. Once the in vivo studies are complete, an in vitro transcription/polyadenylation system will be utilized. It will be determined if the same sequence requirements and regulation can be seen in vitro as in vivo. If this is so, we will begin to fractionate the factors involved in these reactions. An understanding of the regulatory mechanism of polyadenylation will have important implications with respect to not only viral infections but cell growth, differentiation, and development.